1. Field of the Invention
This invention concerns a process for the synthesis of functionalized, water-soluble, macrocyclic complexes of the lanthanides having atomic numbers 57-71, of the actinides having atomic numbers 89-103, and of yttrium(III) having atomic number 39.
This invention also includes processes for the coupling of such complexes to biologically active or biologically compatible molecules, or to biological cells through peripheral pendant substituents having one or more reactive sites. These complexes are useful particularly as reporter molecules in immunoassays, analytical cytology, histological staining, and imaging processes (Leif et al., Clinical Chemistry, 1977, 23, 1492, Development of Instrumentation and Fluorochromes for Automated Multiparameter Analysis of Cells; Leif et al., 1976, Markers for Instrumental Evaluation of Cells of the Female Reproductive Tract: Existing and New Markers, in The Automation of Uterine Cancer cytology, Ed. Wied, Bahr and Barrels pages 313-345).
2. Description of the Prior Art
The complexation of lanthanide ions with organic as well as inorganic compounds and the subsequent use of the resultant complexes in both industrial and biological environments has been previously reported. Such complexes, also referred to as "coordination compounds", are typically formed by the union of a lanthanide ion with appropriate ligands, which can be negatively charged or ionically neutral.
The complexes of the lanthanide(III) ions with unidentate ligands or bidentate chelating ligands are known to be extremely labile, achieving complexation and ligand-exchange equilibrium almost instantaneously. Thus, even very stable complexes of the lanthanide(III) ions and bidentate chelating ligands, e.g. tris(acetylacetonato)-lanthanum(III) exist in solution as a mixture of species, the uncomplexed (solvated) metal ion being present in an appreciable amount. Lanthanide complexes of fused polychelating non-cyclic ligands are somewhat less labile; whereas, those of cyclic ligands are relatively inert.
The stability and lability of the lanthanide(III)-ligand complex can be critical, depending upon the particular use and/or environment. More specifically, the time frame of metal-exchange and ligand-exchange kinetics is a major consideration when such complexes are used as probes in biological systems. In the dilute aqueous solutions or aqueous-organic solutions required for these systems, often involving contact with potentially competing ligands, such complexes, if labile, can dissociate. Where such dissociation does occur, the value of the probe is diminished or lost. Alternatively, vibrational quenching of the fluorescence by interaction with water molecules may occur. For instance as reported by Evangelista, R. A., A New Europium Chelate for Protein Labelling and Time-Resolved Fluorometric Applications, Clinical Biochemistry, 1988, 21, 173-178, the dinegative anionic ligand 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxilic acid (BCPDA) forms a fluorescent complex with europium(III). However, this complex is not detected at concentrations required to detect many analytes of interest which are present in picogram or lower concentrations. Since BCPDA complex of europium(III) ion binds sufficient water to quench the fluorescence, the added inconvenient step of drying the samples must be performed prior to measurement. Furthermore, the quantum efficiency of the BCPDA europium(III) complex is relatively low, so that as reported by Khosravi, M. J. and Diamandis, E. P., Time-Resolved Immunofluorometry of Follitropin in Serum, Clinical Chemistry, 35, No. 1, 181 (1989), it was necessary to increase the sensitivity for analytes which are present in low concentration, by binding the chelate to thyroglobulin as an intermediate step to build a multilayer system.
Soini and Lovgren, Time-Resolved Fluorescence of Lanthanide Probes and Applications in Biotechnology, CRC Critical Reviews in Analytical Chemistry Vol. 18., Issue 2 (1987) pages 105-154; Soini and Hemmila, Fluorescence Spectroscopy Assay Means, U.S. Pat. No. 4,374,120, (1983) have reported on the use of a fluorescent chelate of a lanthanide. The ligand was DTPA (WO Patent 03698, 1984), which forms a strong but nonfluorescent complex with europium(III). This complex was dissociated with acid Hemmila et al. Europium as a label in Time-Resolved Immunofluorometric Assays, Anal. Biochem. 137, 335-343 (1984); and the solubilized europium(III) was complexed with a beta-diketone in a micellar phase. These dissociation-complexation steps resulted in an increase in complexity of the procedure; in a decrease in the concentration of the chelate, which limited the sensitivity of the fluorescence measurement; and in a loss of spatial information due to the separation of the fluorophore from the binding moieties. Because of the separation of the fluorescent tag from the specific binding molecule, this technique is incompatible with immunofluorescence or similar measurements on single cells, or other particles by either flow cytometry or microscopy.
The preparation of inert, kinetically stable, non-functionalized macrocyclic complexes of a limited number of lanthanide(III) ions was first reported in the technical literature by Backer-Dirks et al., J.C.S. Chem. Comm. (1979) 774. The synthesis described in this paper involved a metal templated Schiff-base condensation of 2,6-diacetylpyridine with ethylenediamine in the presence of the nitrate salts of lanthanum(III) and cerium(III). The conditions selected for the condensation were, however, reportedly unsuccessful for the preparation of analogous complexes from the heavier members of the lanthanide series. Because of the high kinetic stability of these complexes and their resistance to dissociation in dilute aqueous solutions, the authors suggested their potential use as aqueous NMR shift reagents.
The synthesis of kinetically stable macrocyclic complexes of the lanthanide(III) ions by metal templated condensation techniques has recently been extended to include all elements within the lanthanide series, except radioactive promethium, .sub.61 Pm.
The following additional references describe known non-functionalized macrocyclic complexes. List A includes publications by at least one of the inventors. List B includes additional publications by others.